Bacteria evolve macroscopic multicellularity by the genetic assimilation of phenotypically plastic cell clustering

Abstract

The evolutionary transition from unicellularity to multicellularity was a key innovation in the history of life. Experimental evolution is an important tool to study the formation of undifferentiated cellular clusters, the likely first step of this transition. Although multicellularity first evolved in bacteria, previous experimental evolution research has primarily used eukaryotes. Moreover, it focuses on mutationally driven (and not environmentally induced) phenotypes. Here we show that both Gram-negative and Gram-positive bacteria exhibit phenotypically plastic (i.e., environmentally induced) cell clustering. Under high salinity, they form elongated clusters of ~ 2 cm. However, under habitual salinity, the clusters disintegrate and grow planktonically. We used experimental evolution with Escherichia coli to show that such clustering can be assimilated genetically: the evolved bacteria inherently grow as macroscopic multicellular clusters, even without environmental induction. Highly parallel mutations in genes linked to cell wall assembly formed the genomic basis of assimilated multicellularity. While the wildtype also showed cell shape plasticity across high versus low salinity, it was either assimilated or reversed after evolution. Interestingly, a single mutation could genetically assimilate multicellularity by modulating plasticity at multiple levels of organization. Taken together, we show that phenotypic plasticity can prime bacteria for evolving undifferentiated macroscopic multicellularity.

Fig. 1

Both E. coli (Gram-negative) and S. aureus (Gram-positive) show the capacity to form phenotypically plastic elongated macroscopic cell clusters.

Fig. 2. Experimental evolution of macroscopic multicellularity.

a A schematic of our experimental evolution workflow. b Clonal phenotypes at the end of experimental evolution after growth under static conditions. Also see Supplementary movies 1 & 3 (for Anc), 5 & 7 (for the S clones), and 6 & 8 (for the R clones). In R1-R5, the habitual salinity tubes were externally perturbed at the end of the growth cycle to disrupt mats formed at the air-liquid interface and show cell clustering (see Supplementary Fig. S4 for the unperturbed tubes).

Fig. 3. The evolution of phenotypic plasticity in cellular morphology.

The arrows point towards the qualitative direction of phenotypic plasticity. The lower and upper box hinges show the 25 and 75% quantiles, respectively; the thick horizontal band represents the median. The lower whisker denotes the smallest observation ≥ lower hinge − 1.5 × interquartile range; the upper whisker represents the largest observation ≤ upper hinge + 1.5 × interquartile range. Two-tailed t-tests (unequal variance across types): *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. See Supplementary Table S1 for statistical details (exact P values). The reversal of the ancestral cell perimeter plasticity (observed in eight out of 10 evolved clones) corresponded to the genetic assimilation of phenotypically plastic cell clustering (compare with Fig. 2b). All the plotted data are provided in the Source Data file. Also see Supplementary Fig. S6 for cell shape plasticity quantified in terms of cellular circularity.

Fig. 4. The genetic basis of assimilated multicellularity.

a Experimental evolution of genetically assimilated multicellularity primarily enriched mutations in genes involved in cell wall assembly. The schematic shows the proteins encoded by the mutated genes in red. The numbers accompanying the mutated proteins represent the number of clones that showed a mutation in a particular protein. Two genes (murF and mppA) showed synonymous mutations. b The location of mutations on the 3D structure of MraY, the protein that mutated in 70% of the sequenced clones. All the mutated regions are located near the periplasmic region of the transmembrane protein.